Prolactin (PRL) is a polypeptide hormone produced by the anterior pituitary. This hormone is important in a number of physiological events, including maintaining normal reproductive functions, osmoregulation, growth, and immunoregulation.
Human prolactin has been characterized as a protein of 199 amino acids and as having a molecular weight of about 23 kDa. Prolactin from other species are reported to have a similar size, and to share a high degree of sequence homology.
For many years, PRL was considered an unmodified polypeptide hormone. It is now clear, however, that posttranslational processing of PRL causes it to be phosphorylated, glycosylated and variously proteolytically cleaved. The phosphorylation of PRL within pituitary cells has also been demonstrated to occur in vivo in the rat, chicken, and cow. Phosphate analysis of purified preparations of PRLs from different species have demonstrated that prolactin from these species are variously phosphorylated with molar ratios of hormone to phosphate of 1.0:0.2 for bovine and rat and 1.0:0.7 for turkey.
Brooks et al., Molecular and Cellular Endocrinology, 99, pp. 301-305 (1994), have reported the isolation of a serine position 90 phosphorylated bovine prolactin species (position 90 relative to the naturally occurring amino acid sequence of bovine PRL), and examined the bioactivity of this molecule relative to native bovine prolactin. The native bovine prolactin was reported to be biologically active for stimulating cell proliferation in the Nb2 rat lymphoma bioassay used by those investigators, however, the serine 90 phosphorylated bovine prolactin was reported to be biologically inactive in the same bioassay.
Earlier functional studies have examined another phosphorylated prolactin species from the rat, this molecule being phosphorylated at serine residue 177. It was reported in that study that this mono-phosphorylated species of PRL (phosphoserine at position 177 of native rat prolactin amino acid sequence) acted as an antagonist to native, non-phosphorylated PRL in two cell systems (pituitary tumor cells, specifically GH3 cells that are PRL-producing rat pituitary tumor cell); and T lymphoma cells (Nb2 cell bioassay). Of significance, the serine 177 mono-phosphorylated-rat species of PRL was observed to act as an antagonist to non-phosphorylated prolactin, and to inhibit cell proliferation of Nb2 cells. Wang et al., Endocrinology, 133, pp. 2156-2160 (1993). The non-phosphorylated PRL was observed to promote cell proliferation of these prolactin dependent tumor cell lines. These observations stand in contrast to the lack of biological activity reported with the phosphorylated PRL species described in Brooks et al. (1994).
Native rat PRL is subject to ready phosphorylation in vivo. Those of skill in the art hold the view that these phosphorylated forms of PRL can be hydrolyzed by serum phosphatase to form non-phosphorylated prolactin, thus adding to the pool of non-phosphorylated PRL. As noted above, native, non-phosphorylated rat PRL has been reported to stimulate tumor cell growth. Thus, non-phosphorylated prolactin pools would be undesirable in the treatment of prolactin dependent tumors. This would render therapies with phosphorylated prolactin disadvantageous, as it may actually function to increase the proliferation of prolactin dependent tumors by supplying more of the prolactin species that binds prolactin receptor and turns on prolactin secretion and tumor growth. The half-life of phosphorylated prolactin is also relatively short, making it undesirable for use as a therapeutic. Recognition of this phenomenon makes the use of mono-phosphorylated PRL forms undesirable, as it may potentially worsen some conditions rather than provide a treatment.
U.S. Pat. No. 4,725,549, relates to a description of human and rat prolactin and preprolactin cloned genes. Paris et al., Biotechnology & Applied Biochemistry, 12, pp. 436-449 (1990) have described a recombinant human PRL that includes an additional methionine group at the amino terminal end of the molecule. These recombinant PRL species were observed to comigrate with human PRL on an SDS-polyacrylamide gel, and to have parallel dose response activity for stimulating Nb2 cell proliferation. The hPRL cDNA coding sequence reported by Cooke et al., Journal of Biological Chemistry, 256, pp. 4006-4016 (1981), and the T7 expression vector of Studier and Moffat, Journal of Molecular Biology, 189, pp. 113-130 (1986), was employed in preparing recombinant hPRL in a bacterial E. coli system. This technique provided a method for producing large quantities of recombinant non-phosphorylated PRL. However, a recombinant phosphorylated PRL, or a recombinant protein or peptide that mimics the prolactin antagonistic activity of phosphorylated PRL, has not before the present disclosure been described.
Abnormal levels of prolactin or abnormalities in the production of physiological concentrations of this hormone have been associated with several pathologies, including prolactin-dependent tumors. LHRH-like peptide prolactin antagonists have been proposed in the treatment of several specific pathologies, including hyperprolactinemia, miscarriage, precocious puberty and ovulation irregularities. For example, an LHRH C-terminal peptide has been described for use as a prolactin antagonist in the inhibition of prolactin-responsive tumors (U.S. Pat. No. 5,075,224).
A need continues to exist in the art for more stable and effective forms of non-LHRH like prolactin antagonists for therapeutic use. These molecules will preferably be capable of functioning as effective prolactin antagonists that compete with native prolactin for prolactin receptor sites, and which at the same time are not subject to dephosphorylation, thereby avoiding an increase in the pool of dephosphorylated prolactin. Such preparations could be used to control prolactin secretion and prolactin dependent tumor cell proliferation, as well as provide an improved treatment for other pathologies associated with abnormal prolactin regulation.